The N terminus of human INCENP (GFP-INCENP^1-58) targets to the centromere. HeLa S3 cells were transfected with INCENP^1-58 or INCENP^59-919 N-terminally fused to GFP. (A) GFP-INCENP^1-58 targets to the centromere in prometaphase and metaphase (independently of its expression level) but remains cytoplasmic in anaphase and telophase. (B) A GFP-INCENP construct lacking the first 58 amino acids (GFP-INCENP^59-919) does not localize to any defined cellular structure during mitosis. Bar, 10 μm.

Borealin and Survivin form oligomeres at the centromere. (A) HeLa S3 cells were transiently transfected with the indicated constructs, arrested in prometaphase by treatment with nocodazole for 16 h, and then processed for indirect immunofluorescence analysis using anti-Flag or anti-Myc antibodies (green), respectively, and anti-Aurora B antibody (red). Bar, 10 μm. (B) Myc- or Flag-tagged CPC constructs were cooverexpressed in HeLa S3 cells followed by Myc and Flag pull-downs and Western blotting with anti-Flag or anti-Myc antibodies. Both Borealin and Survivin form oligomeres in this assay (compare lanes 3 and 4 of I, II, and III).

siRNA suppression and simultaneous rescue of INCENP by ectopic expression. (A) HeLa S3 cells were treated with siRNA oligonucleotides targeting the 3′ UTR of INCENP, Aurora B, Borealin, or Survivin transcripts, fixed, and then costained with antibodies against INCENP and Aurora B (INCENP- and Aurora B-RNAi, top) or Borealin and Survivin (Borealin- and Survivin-RNAi, bottom). Mitotic cells are indicated by arrows. Bar, 10 μm. (B) Cell extracts from cells depleted for the different passenger proteins by treatment with corresponding siRNA oligonucleotides for 36 h were analyzed by Western blotting. The blots were reprobed with antibodies against CENP-A to demonstrate equal loading (bottom row). (C) HeLa S3 cells treated with the indicated 3′ UTR siRNA oligonucleotides targeting chromosomal passenger proteins for 36 h were stained with antibodies against Aurora B. One hundred cells were assessed in each case for the presence of Aurora B centromere staining. (D) HeLa S3 cells were treated with 3′ UTR siRNA oligonucleotides targeting INCENP and simultaneously transfected with GFP-INCENP^full-length. Transfected cells were analyzed for the presence of the other chromosomal passenger proteins or phospho-CENP-A staining as a readout for Aurora B activity. All images shown are representatives of three independent experiments. Bar, 10 μm.

Centromere targeting of the CPC is independent of CENP-A and hMis12. (A) Lamin A (control) and CENP-A were knocked down by siRNA treatment. HeLa S3 cell lysates were probed by Western blotting using the indicated antibodies. (B) CENP-A or hMis12 siRNA-treated HeLa S3 cells were analyzed by immunofluorescence for the presence of CENP-A, Borealin, or Aurora B. Bar, 10 μm.

In vivo targeting of Borealin and Survivin to the centromere requires the presence of their binding partners. (A) HeLa S3 cells were treated with 3′ UTR siRNA oligonucleotides targeting Borealin and simultaneously transfected with Myc-Borealin (see Figure 5C for RNAi efficiency). Transfected cells were analyzed for the presence of the other chromosomal passenger proteins. (B and C) Cells were treated as in A except that INCENP (B) or Survivin (C) were knocked down in parallel with Borealin. (D) HeLa S3 cells were treated with siRNA oligonucleotides targeting the 3′ UTR of the Survivin transcript (see Figure 5C for RNAi efficiency) and simultaneously transfected with Myc-Survivin. Transfected cells were analyzed for the presence of INCENP, Borealin, and phospho-CENP-A by indirect immunofluorescence. (E and F) HeLa S3 cells were treated as described for D but in addition to Survivin, either INCENP (E) or Borealin (F) was knocked down. All immunofluorescence images are representatives of three independent experiments. Bar, 10 μm.

INCENP^1-58, Borealin, and Survivin form a complex. (A) pFBT9-Aurora B (I and IV), pFBT9-Borealin (II and V), and pFBT9-Survivin (III) were tested against different fragments of INCENP (I–III) and full-length Borealin or Survivin (IV and V) in pACT2 by two-hybrid analysis. INCENP fragments were created based on a coiled-coil prediction tool (Berger et al., 1995). INCENP^1-58 could not be used in this assay because it was self-activating. −LW indicates plates lacking leucine and tryptophane, whereas QDO indicates plates lacking leucine, tryptophane, histidine and adenine. (B) MBP (lanes 1 and 2) or MBP-INCENP^1-58 (lanes 3–5) was incubated with His-Borealin immobilized on Ni-NTA agarose (lanes 2 and 4) or Ni-NTA agarose alone (lane 5). Input and protein bound to the beads were analyzed by SDS-PAGE. (C) MBP (lanes 1, 4, 8, and 9), MBP-INCENP^1-58 (lanes 2, 5, 6, 10, and 11), and MBP-Borealin (lanes 3, 7, and 12) were incubated with GST (lanes 4–7) or GST-His-Survivin immobilized on glutathione-Sepharose (lanes 8–12). Zn²⁺ (20 mM) was added to the reactions where indicated (lanes 6, 9, and 11).

INCENP^1-58, Borealin, and Survivin form a ternary complex in vivo. (A) Different fragments of INCENP fused to GFP were transfected into HeLa S3 cells and precipitated from mitotic lysates with sheep anti-GFP antibodies. Coprecipitating passenger proteins or the control centromere/kinetochore proteins CENP-A and Hec1 were visualized by Western blotting. Asterisks indicate GFP constructs. The anti-GFP blot was cut into two halves to remove the strong signal of the immunoglobulin heavy chain.

GFP-INCENP^1-58 targets to the centromere in the absence of endogenous INCENP and restores Survivin and Borealin but not Aurora B staining. (A) HeLa S3 cells were treated with 3′ UTR siRNA oligonucleotides targeting INCENP and simultaneously transfected with GFP-INCENP^1-58. Transfected cells were analyzed for the presence of the other chromosomal passenger proteins or phospho-CENP-A staining as a readout for Aurora B activity. (B) HeLa S3 cells were transfected with the indicated constructs in pcDNA4/TO-EGFP encoding puromycin resistance and treated with the indicated siRNA oligonucleotides for 36 h. Twenty-four hours before harvesting, the cells were treated with 2 μg/ml puromycin. Asterisks indicate GFP constructs. (C) INCENP siRNA-treated HeLa S3 cells transfected with INCENP^59-919 were treated as in A and costained for pCENP-A. (D) Recruitment of BubR1 to the kinetochore as a readout for normal chromosomal passenger function was visualized by immunofluorescence in INCENP knockdown cells transfected with either GFP-INCENP^full-length (top) or GFP-INCENP^1-58 (bottom). (E) The percentage of transfected cells with properly aligned metaphase plates (left), prometaphase-like chromosome configurations (middle), or with metaphases with uncongressed chromosomes (right) was quantitated in cells treated with 3′ UTR siRNA oligonucleotides against INCENP and transfected with either GFP-INCENP^full-length (gray columns) or GFP-INCENP^1-58 (black columns). (F and G) HeLa S3 cells were treated as in A but in addition to INCENP, Survivin (F) or Borealin (G) was simultaneously knocked down. All immunofluorescence images are representatives of three independent experiments.

Borealin binds to double-stranded DNA in vitro and recruits Survivin and INCENP^1-58 to DNA. (A) Histone H3, His-Survivin, His-Borealin, MBP-Borealin, MBP-INCENP^1-58, MBP alone, GST-Cdc20, and His-Plk1 were tested for DNA-binding activity by incubation with calf-thymus DNA-cellulose. Input (lanes 1, 3, 6, 8, 10, 12, 14, and 16) and proteins bound to the cellulose (lanes 2, 4, 5, 7, 9, 11, 13, 15, and 17) were analyzed by SDS-PAGE. Only histone H3 (lane 2), His-Borealin (lane 7), and MBP-Borealin (lane 9) bound to the DNA cellulose. (B) His-Borealin was incubated with DNA-cellulose as in A in the presence of increasing NaCl concentrations. (C) His-Borealin, His-Survivin, and MBP-INCENP^1-58 were mixed at equimolar concentrations and incubated with calf-thymus DNA-cellulose as described above. In the presence of His-Borealin, His-Survivin and MBP-INCENP^1-58 were found in the DNA-binding fraction (lane 4).

Model for centromere targeting of the CPC. (A) Our data suggest that Survivin and Borealin, both forming higher molecular order structures at the centromere, bind to the first 58 amino acids of INCENP. Aurora B, in contrast, associates with the C-terminal IN-box of INCENP. We propose that Borealin targets the CPC to the centromere by binding to DNA directly (dashed arrow) but can only do so when present within a functional subcomplex. Aurora B kinase is not involved in this process. (B) The centromere recruitment of the CPC is independent of the CENP-A and the hMis12 assembly pathways. Both pathways are required for the targeting of CENP-I and CENP-H (not depicted); in addition, CENP-A recruits the Hec1/Nuf2-complex. The CPC recruits MCAK to the inner centromere and checkpoint proteins to the kinetochore.